The polymer particles having sub-micron to micron size are used in various applications such as an organic pigment, toner grains, a spacer for liquid crystal, latex microparticles and a filler for ion-exchange liquid chromatography. Out of these, the application to the filler for ion-exchange liquid chromatography has been attracting attention in recent years.
The ion-exchange liquid chromatography method has been known as a highly effective method for separation analysis of various living body-related substances. Especially, an application to a method for analyzing glycosylated hemoglobins (hereinafter, also referred to as hemoglobin A1c) has been attracting attention in recent years.
Hemoglobin A1c is a form of hemoglobin that has blood sugar chemically bound to an N-terminus of a β chain. A proportion of hemoglobin A1c in hemoglobins, that is, a proportion of hemoglobin A1c in a sum of hemoglobin A1c and non-glycosylated hemoglobin is considered to reflect an average blood sugar level in a period of one to two months. Therefore, a hemoglobin A1c value (%) which represents the proportion of hemoglobin A1c has been increasingly used as an indicator for diabetes diagnosis in place of a blood sugar level which may show temporary and rapid fluctuation.
Polymer microparticles to be used in an aqueous medium is required to have a low degree of swelling in the aqueous medium for the purpose of suppressing deformation due to environmental change in the aqueous medium, and also to have the particle surfaces with a high hydrophilicity for the purpose of increasing dispersibility and the like. Polymer microparticles to be used as a filler for ion-exchange liquid chromatography in the ion-exchange liquid chromatography method, in particular, are required to have a significantly low degree of swelling for the purpose of suppressing pressure fluctuation in a column in order to accelerate equilibration.
In order to obtain the polymer microparticles having a low degree of swelling in an aqueous medium, as a conventionally well-known method, a large amount of hydrophobic crosslinking monomers are used to raise a degree of crosslinking.
However, there has been a problem that the microparticles containing the hydrophobic crosslinking monomers have a low dispersibility in an aqueous medium due to its high hydrophobicity of the surface. Furthermore, in case of using microparticles of this kind as a filler for liquid chromatography, there has also been a problem that non-specific adsorption occurs upon its contact with a living body sample such as protein.
Since a hydrophobic interaction is considered to cause the non-specific adsorption, the filler for ion-exchange liquid chromatography is required to have the surface with as high a hydrophilicity as possible.
As a method for increasing the hydrophilicity of the filler for ion-exchange liquid chromatography, examples thereof include a method in which base microparticles of the filler for ion-exchange liquid chromatography are allowed to contain a large amount of hydrophilic monomers, and the like.
However, the large content of the hydrophilic monomers leads to an increase in hydrophilicity of the inside of the filler for ion-exchange liquid chromatography. As a result, the filler for ion-exchange liquid chromatography has a decreased mechanical strength, leading to the following problems: the filler for ion-exchange liquid chromatography can not be used for high-speed separation; measurement accuracy decreases due to swelling of the filler for ion-exchange liquid chromatography itself; and the like.
As a method for solving these problems, for example, there has been known a filler having an ion exchange group introduced into a base material made of a siliceous compound, and a filler obtained by a reaction of crosslinking particles made of an organic synthesis polymer with a compound containing the ion exchange group (Patent Document 1 etc.). Further, there has been known a filler obtained by a reaction of the crosslinking monomer and a compound containing an ion exchange group (Patent Document 2 etc.).
Furthermore, Patent Document 3 discloses a coated polymer microparticle having a layer of hydrophilic polymers formed on a surface of a hydrophobic crosslinked polymer microparticle. The hydrophobic crosslinked polymer microparticle is strongly crosslinked by hydrophobic crosslinking monomers contained therein, so that the hydrophobic crosslinked polymer microparticle has a high mechanical strength and shows reduced swelling. Moreover, it concluded that it is possible to prevent non-specific adsorption of an object to be analyzed and the like, and suppress a degree of swelling while maintaining its hydrophilicity, by forming the layer of the hydrophilic polymer with a thickness of 1 to 30 nm.
However, in practical use, it has been difficult for the hydrophilic polymer with a thickness within the above-mentioned range to prevent exposure of the hydrophobic crosslinked polymer microparticle. As a result, it has been impossible to sufficiently prevent the non-specific adsorption caused by a hydrophobic interaction.
In particular, in case of measuring a substance used for a clinical examination and the like such as glycosylated hemoglobin, since a significantly higher level of measurement accuracy is required, it is necessary to prevent non-specific adsorption caused by the hydrophobic interaction as much as possible.
On the other hand, Patent Document 4 discloses a filler obtained by hydrophilizing filler particle surfaces having an ion exchange group, specifically a hydrophilized filler for ion-exchange liquid chromatography obtained by adsorption of compounds having a hydrophilic group, such as protein, on the filler particle surfaces having the ion exchange group. In the filler for ion-exchange liquid chromatography of this kind, while a base material does not swell or shrink, the hydrophilic surface enables effective prevention of non-specific adsorption of protein and the like. However, in the case where hydrophilic compounds are fixed by the physical adsorption as described above, although the high performance is demonstrated in an early stage of use, long-term use causes detachment of the hydrophilic compounds from the filler particle surfaces, leading to a problem that retention times and measured values may be varied. Moreover, there is also a problem that lot to lot variation in the hydrophilic compounds to be used for the adsorption causes variations in retention times and measured values.
Patent Document 1: Japanese Kokai Publication No. Hei-1-262468
Patent Document 2: Japanese Kokoku Publication No. Sho-63-59463
Patent Document 3: Japanese Kokoku Publication No. Hei-8-7197
Patent Document 4: Japanese Kokai Publication No. 2001-91505